Secretory Pathway Calcium-ATPases (SPCA) are a newly defined family of ion pumps that transport calcium and manganese into the lumen of the Golgi apparatus where they are essential for sorting, processing, and glycosylation of proteins. The first member of this family, named PMR1, was described in Saccharomyces cerevisae, and more recently, two mammalian homologues of PMR1, SPCA1 and SPCA2, have been identified. Mutations in hSPCAl cause Hailey Hailey disease, a debilitating disorder characterized by severe ulceration of the skin, thought to result from dysregulation of cellular calcium. Excess manganese is deposited in the brain and leads to Parkinsonism. The SPCA have been implicated in diverse physiological processes, ranging from manganese detoxification in the liver, calcium transport across intestinal epithelia, and milk production by the mammary glands, although there is little molecular evidence for their specific roles. This proposal combines three parallel approaches to investigate the SPCA: biochemical studies using purified proteins or Golgi membranes, cell biological studies in polarized cultured mammalian cells, and large scale phenomic analysis of ion homeostasis in a model organism. In previous studies, we have defined the transmembrane helices and residues critical for ion transport and have identified a role for helix packing in determining ion selectivity. In Aim 1 of this proposal, we will shift our focus to understanding the ion binding and modulatory role of EF motifs in the cytoplasmic N-terminal domain. We have new evidence for trafficking of the pumps between the Golgi stacks and a novel, vesicular compartment in polarized cell models of hepatocytes and enterocytes. In Aim 2, we will determine if trafficking is ion-dependent and related to transcellular transport, and whether specific retrieval or PDZ-binding motifs at the C-terminus are important for localization. Gene knockdown approaches will be used to evaluate the isoform-specific contributions of the two SPCA pumps in the enterocyte model. Finally, we propose to use the yeast model organism for high resolution phenomic analysis that will identify new genes and pathways associated with the cellular function of the SPCA pumps (Aim 3). Taken together, this proposal lays the foundation for understanding the role of this novel family of transporters at the molecular, cellular and physiological level.